Conventional adenovirus (Ad) gene-delivery vectors are based on replacement of early regions of the viral genome with an expression cassette coding for a gene of interest. Unfortunately, Ad vectors have drawbacks that limit their usefulness for many applications. First, the cloning capacity of these vectors is limited to 8-10 kb. Second, despite deletion of the E1 region, leaky expression of immunogenic viral proteins occurs in vivo, which leads to a host immune response and elimination of gene expression from transduced tissues. Gutted, or helper-dependent, adenoviral vectors may overcome these drawbacks. Gutted vectors contain cis-acting DNA sequences necessary for viral replication and packaging, but usually do not contain viral coding sequences (See U.S. Pat. No. 6,083,750, incorporated by reference). These vectors can accommodate up to 36 kb of exogenous DNA and are unable to express viral proteins. Gutted vectors are produced by replication in the presence of a helper virus, which provides all necessary viral proteins in trans. Since the viral proteins act to replicate both gutted and helper genomes, gutted adenovirus particles are prepared as a mixture with helper virions, though selection against helper virus packaging can reduce this contamination. Particles containing gutted viral genomes, rather than helper genomes, are subsequently purified on the basis of their lower density.
Generally, the starting point for production of a gutted virus is plasmid DNA. The plasmid contains the viral inverted terminal repeats (ITRs), the viral packaging signal, and exogenous DNA to be carried by the gutted virus. To increase production of gutted virus, most investigators linearize the gutted viral plasmid (some systems require the ligation of viral ITRs after linearization). The plasmid DNA is co-introduced with helper sequences into a cell line that can replicate the helper virus, normally 293 cells. Replication of the helper virus eventually causes lysis of the cells with the lysate containing a large number of helper virions and a comparatively small number of gutted virions.
To increase the number and proportion of gutted virions in the lysate, the initial mixture is generally serially passaged. Helper-dependent Ad vectors are usually propagated with constant selective pressure against helper virus packaging. During early passages, selection allows for gradual improvement in the ratio of gutted to helper virus. At the last passage selection removes the majority of helper virus before further purification. Unfortunately, growth of vector stocks under selective pressure can lead to rearrangement of helper and gutted viruses.
The production of gutted virus particles from plasmid DNA in the first step of gutted vector production is so inefficient that titers of less than 100 particles per milliliter have been reported. In some cases no gutted virions can be detected until at least one serial passage has been performed. What is needed is methods and compositions for faster, higher titer and higher purity production of adenovirus vectors.